Quality of eggs from Lohmann Brown Classic laying hens fed black
soldier
fly meal as substitute for soya bean
G. Secci
1, F. Bovera
2, S. Nizza
2, N. Baronti
1, L. Gasco
3, G. Conte
4, A. Serra
4, A. Bonelli
1and
G. Parisi
1†1Department of Agri-Food Production and Environmental Sciences, Section of Animal Sciences, University of Firenze, via delle Cascine 5, 50144 Firenze, Italy; 2Department of Veterinary Medicine and Animal Production, University of Napoli Federico II, via F. Delpino 1, 80137 Napoli, Italy;3Department of Agricultural,
Forest, and Food Sciences, University of Torino, largo Braccini, 2, 10095 Grugliasco, Torino, Italy;4Department of Agriculture, Food, and Environment, University
of Pisa, via del Borghetto 80, 56124 Pisa, Italy
(Received 22 June 2017; Accepted 27 November 2017)
Soya bean is the main protein source in poultry feed but rising prices make an alternative protein source necessary. Insects, such as the black soldierfly (Hermetia illucens), may be an attractive solution for hens, although little information is available on their effect on egg quality. The present study aims tofill this gap by testing the effect of 100% replacement of soya bean with
H. illucenslarva meal in the diet of Lohmann Brown Classic laying hens for 21 weeks. At the end of the trial, the eggs were characterized for parameters such as weight, colour, proximate composition of albumen and yolk, and content of carotenoids, tocopherols and cholesterol. The fatty acid profile of yolks was also determined. Hens fed the insect-based diet produced eggs (HIM group) with a higher proportion of yolk than the group fed the soya bean-based diet (SBM group). HIM was associated with redder yolks (red index 5.63v. 1.36) than SBM. HIM yolks were richer inγ-tocopherol (4.0 against 2.4 mg/kg), lutein (8.6 against 4.9 mg/kg),β-carotene (0.33 against 0.19 mg/kg) and total carotenoids (15 against 10.5 mg/kg) than SBM yolks. The fatty acid composition of HIM yolks was almost identical to that of SBM yolks. Finally, HIM yolks contained 11% less cholesterol than SBM yolks. These results suggest thatH. illucenslarva meal is a suitable total substitute for soya bean meal in the diet of Lohmann Brown Classic laying hens. A sustainable alternative to the plant protein source therefore seems feasible.
Keywords:Hermetia illucens, egg, cholesterol, carotenoids, tocopherol
Implications
The need for alternative protein sources in livestock production is a crucial topic. The high prices of raw materials used in diet formulation, as well as public opinion regarding the environ-mental sustainability of animal production are prompting research into new protein sources. The results of the present study show that total substitution of soya bean meal with black soldierfly larva meal in the diet of Lohmann Brown Classic laying hens does not alter overall egg quality and improves parameters such as yolk weight and carotenoid content.
Introduction
It is widely accepted that by 2050 the world will host 9 billion people, prevalently concentrated in regions subject to food insecurity. To accommodate this population, current
animal-derived product supply will need to almost double. Eggs are an inexpensive source of high-quality protein, essential vitamins and minerals (Mirandaet al., 2015). The global population of laying hens grew by 3.3 billion between 1993 and 2013. Global egg production increased by 30.2 million tons in the same period, reaching 68.3 million tons in 2013 (Leeson, 2010). The United Nations’ Food and Agriculture Organization estimates that egg production will reach 89 million tons in 2030 (faostat3.fao.org/home/E).
Appropriate nutrition of hens is a pre-requisite for optimal egg production. Nutritionists design diets to meet laying hens’ energy, protein, mineral and vitamin requirements which depend on maintenance, BW and level of egg production. Specific calcium, choline, vitamin A and vitamin D require-ments have been demonstrated fundamental for hens (Leeson, 2010). In developing countries, protein-rich feed ingredients are generally plant-based, including oil cake, leguminous seeds such as soya beans, and grain such as corn. However, the price of soya bean meal rose to 389 US dollars per metric ton (http://ycharts.com/indicators) in April 2017. Feed price
†E-mail: giuliana.parisi@unifi.it
has a large impact on egg production costs, amounting to 55% to 60% in cage housing systems (International Eggs Commission (IEC), 2015). Chronic shortages of feed resources due to land scarcity and climate change could also have profound implications for feed and food production. Finding alternative sustainable feed options is therefore an urgent priority.
Insects are an increasingly attractive feed resource (Makkaret al., 2014) for chickens and hens (Biasato et al., 2016); indeed, insects are a natural food source for poultry. Their nutritional value has been well documented for larvae and/or prepupae of the moth (Cirina forda), black soldierfly (Hermetia illucens), housefly maggot and mealworm (Tenebrio molitor) raised on different substrates, such as rotting fruit and vegetables, coffee bean pulp, distillers grains,fish offal and particularly animal manure and human excreta (van Huiset al., 2013; Makkaret al., 2014). Among the major insect species studied, we chose the black soldier fly as total soya bean meal substitute in this study because besides other main constituents, it is a good source of Ca (50 to 80 g/kg dry matter) and P (6 to 15 g/kg dry matter) (Gutiérrez et al., 2004; St-Hilaire et al., 2007) which are important for layers. The aim of the present study was to determine the effect on egg nutritional composition of completely replacing soya bean meal in hen feed with black soldierfly (H. illucens) meal.
Material and methods
Experimental design
The birds were treated humanely according to the principles of the EC Directive 2010/63 on the protection of animals used for experimental and other scientific purposes. The experimental procedures were approved by the Ethical Animal Care and Use Committee of the Department of Veterinary Medicine and Animal Production of the University of Napoli Federico II (Italy) (prot. N. 2017/0017676).
Lohmann Brown Classic laying hens were raised on a private farm in Southern Italy. A total of 108, 24-week-old hens (average live weight 1.78 ± 0.15 kg) were divided into two groups (54 hens/group). Up to 45 weeks of age (along an experimental period of 21 weeks), the hens were housed in the same building in modified cages (800 cm2/hen), under controlled conditions of temperature and humidity. The hens of each group were distributed in three cages (18 hens/cage) and each cage was divided by two internal transects into three equal areas, to obtain nine replicates of six hens per group. The two groups were fed two isoproteic and isoenergetic diets, differing in the ingredients used as main protein source. The control diet (C) was based on soya bean meal and was administered to the control group (SBM); the experimental diet (H) was based on defatted meal made fromH. illucens
larvae (Hermetia Deutschland GmbH & Co, Baruth/Mark, Germany), which completely replaced soya bean meal, and was administered to the experimental group (HIM). The list of ingredients and the chemical nutritional characteristics of diets C and H are summarized in Table 1. Metabolizable energy of
the diets was calculated according to NRC (1994) procedure of estimation and the values for the apparent metabolizable energy for the insect meal used in the present trial were obtained from the studies of De Marcoet al. (2015) in broilers. Fatty acid (FA) composition and cholesterol, tocopherol and carotenoid content of C and H are reported in Table 2 together with the profile ofH. illucenslarvae, obtained by the methods described below. The diets, formulated to meet hen require-ments according to the Lohmann Brown Classic Management Guide, and fresh water were administered each dayad libitum. The recorded feed intake was 125.13 and 108.04 g/day per hen for SBM and HIM, respectively (as reported by Marono
et al., 2017). The dark-light cycle was 9 : 15 h. After 2 weeks of adaptation to the new diets (starting 26 weeks after onset of deposition), collection of eggs was begun.
Egg quality determination
A total of 87 eggs (44 for SBM group and 43 for HIM) were collected on eight different sampling days during the whole experimental period (21 weeks) and frozen until analyses. A 24 h defrosting time at 4°C was necessary in order to conduct physical and chemical analyses. In all, 42 eggs (21 for each experimental group) were randomly allotted for Table 1Formulation (g/kg) and chemical nutritional characteristics (g/kg) of the diets used in the trial
Soya bean meal diet (C)
H. illucens meal diet (H) Ingredients (g/kg)
Maize 583.0 653.0
Soya bean meal 235.0 –
Insect meal – 170.0
CaCO3 80.0 80.0
Dehulled sunflower meal 50.0 50.0
Vegetable oil 15.0 10.0 Minimum vitamin1 10.0 10.0 Monocalcium phosphate 5.0 5.0 Salt 2.0 2.0 Chemical nutritional characteristics (g/kg)2 Dry matter 905.0 901.0 CP, as fed 179.0 181.0
Crudefibre, as fed 41.4 39.6
Ether extract, as fed 42.6 43.3
Ash, as fed 142.0 142.0
ME (kcal/kg) 2745 2780
ME= calculated metabolizable energy.
Soya bean meal based (control diet, C) andH. illucens-based meal (H).
1Per kilogram of diet: vitamin A (retinyl acetate) 20 000 IU, vitamin E (dl-α
-tocopheryl acetate) 80 mg, vitamin D3 (cholecalciferol) 6000 IU, vitamin B1
(thiamine monophosphate) 3 mg, vitamin B2 (riboflavin) 12 mg, vitamin B6
(pyridoxine hydrochloride) 8 mg, vitamin B12 (cyanocobalamin) 0.04 mg,
vitamin K3(menadione) 4.8 mg; vitamin H (D-biotin) 0.2 mg, vitamin PP (nicotinic
acid) 48 mg, folic acid 2 mg, calcium pantothenate 20 mg, manganous oxide 200 mg, ferrous carbonate 80 mg, cupric sulphate pentahydrate 20 mg, zinc oxide 120 mg, basic carbonate monohydrate 0.4 mg, anhydrous calcium iodate 2 mg, sodium selenite 0.4 mg, choline chloride 800 mg, 4-6-phitase 1800 FYT,
DL-methionine 2600 mg, canthaxanthin 8 mg.
the evaluation of weight, and egg, yolk and albumen quality for morphological and chemical traits, whereas the other 45 eggs were utilized for other analyses, not considered here. Whole, shell, yolk and albumen weights were recorded. Yolk pH was measured at two points using a pH-meter (Columbus, OH, USA). Yolk colour measurement was performed with a Dr Lange Spectro-colour® colorimeter (Keison International Ltd., Chelmsford, Essex, UK) equipped with Spectral qc 3.6 software, according to the Commission Internationale de L’Eclairage (CIE) system (CIE, 2004) and expressed as lightness (L*), redness index (a*) and yellowness index (b*). Yolk diameter was measured manually with a calliper.
Samples of yolk and albumens were characterized chemi-cally for moisture, CP (N× 6.25) and ash content according to methods of the Association of Official Analytical Chemists (AOAC, 2012). Total lipid content of samples was determined according to Folch et al. (1957), and FAs in lipid extract were trans-esterified to methyl esters by base-catalyzed trans-esterification (Christie, 1982). Fatty acid composition was determined by gas chromatography using a Varian GC 430 gas chromatograph (Varian Inc., Palo Alto, CA, USA). Fatty acids were quantified via calibration curves, using tricosanoic acid (C23:0, 0.4 mg/ml) (Supelco, Bellefonte, PA, USA) as internal standard. Fatty acids were identified and then quantified via calibration curves, using tricosanoic acid (C23:0; 0.4 mg/ml) as internal standard.
Yolk cholesterol content was determined. Briefly, 0.2 ml of lipid extract was spiked with 0.5 ml 5α-cholestane (0.2 mg/ml prepared in chloroform) (Supelco) as internal standard. After solvent evaporation, 5 ml KOH (0.5 M in methanol) was added and left in a 95°C bath for 40 min to promote lipid saponification. Finally, 4 ml distilled water and 2 ml n-hexane were added. The upper phase was then transferred directly to a vial for GC analysis using the Varian GC 430 gas chromatograph (Varian Inc.), equipped with aflame ionization detector and a Supelco SACTMfused silica capillary column (30 m× 0.25 mm i.d., 0.25-μm film; Supelco), purchased from Agilent Technologies (Santa Clara, CA, USA). A sample of 1μl was injected with a 1 : 100 split ratio at 300°C. Oven temperature was programmed to rise from 130°C to 290°C in 8 min (20°C/min) then remain at 290°C for 11 min. The detector was set at 300°C. Helium was used as carrier gas at a constantflow of 1.3 ml/min.
The carotenoid and vitamin E content of feeds and yolks was determined in the lipid extracts, adjusting the methods previously described by Majchrzaket al. (2006) and Prates
et al. (2006). Lipid extracts were saponified by the method
already described for cholesterol but without the warm bath. Indeed, saponification was achieved keeping the samples at room temperature overnight. Unsaponified matter was then resuspended in 200μl chloroform/methanol (1 : 1) solution. Finally, 20μl of each sample was quantified using a Prostar HPLC instrument (Varian Inc.) with UV-DAD and a C18reverse
phase column (ChromSep HPLC Columns SS 250 mm× 4.6 mm with ChromSep guard column Omnispher 5 C18). The
mobile phases were (A) methanol : acetonitrile : water (5 : 85 : 10) and (B) methanol : ethylacetate (70 : 30). Flow
conditions were 90 : 10 of mobile phases A and B, respecti-vely, at 1 ml/min for 18 min, followed by 50 : 50 (1 ml/min) for 2 min, followed by 0 : 100 at 1.5 ml/min for 10 min. Carotenoids (lutein, zeaxanthin andβ-carotene) were detected at 450 nm, vitamin A (retinol) at 325 nm and tocopherols (α,γandδ) at 292 nm. All molecules were quantified using an external calibration curve with concentrations ranging from 0.005 to 0.17µg/ml, 0.02 to 0.5 µg/ml and 0.01 to 0.97µg/ml for carotenoids, retinol and tocopherols, respecti-vely. Results were expressed as mg/kg total lipids.
Statistical analysis
To test the effect of the diet, the data were analysed by one-way ANOVA by PROC GLM of SAS statistical software (SAS, 2004), with the following linear model:
Yiz= μ + αi+ eiz
where Yizis the dependent variable of thezthobservation;
µthe overall mean;αithefixed effect of theithdiet (i= C, H)
and eiz the random error. Each egg from the two
experi-mental groups was considered a biological replicate. Results
Chemical characterizations of H. illucenslarva meal and of the two experimental diets (C and H) are reported in Table 2. Linoleic (18:2n-6, LA), oleic (18:1n-9) and palmitic (16:0) acids were the major FAs in both feeds, amounting to 70% and 62% of total FAs in C and H, respectively. The H diet was mostly distinguished by a higher content of lauric acid (12:0) than the control diet, which in turn was higher in 18:2n-6. The H diet was 8% higher in saturated fatty acids and 6% lower in polyunsaturated n-6 fraction (PUFA n-6) than the control diet, while PUFA n-3 was slightly higher in H than C. Cholesterol was not detected in H. illucens larva meal. Comparing vitamin and carotenoid content, H was lower in total tocopherols than the control diet, with half the amount ofα-tocopherol, despite the high content of this vitamin in the insect meal.
Regarding retinol content,H. illucensmeal did not contain vitamin A, while diet H had higher retinol than the control diet. Diet H was also four times richer in total carotenoids than diet C, especially lutein which was more than three times richer than in C.
The effects of the diets on egg characteristics of Lohmann Brown Classic hens are shown in Table 3. Whole egg weights, shell weights and yolk diameter were not signi-ficantly affected by diet, whereas albumens from the HIM group were significantly lighter and the yolks from this same group of hens slightly heavier than those from the SBM group. The ratio yolk to albumen was 0.38 and 0.42 in SBM and HIM, respectively. The yolks of eggs from the HIM group were redder (P< 0.01) than those of the control group. The
chemical composition of yolks and albumens was unaffected by dietary treatments (Table 4). With regard to tocopherols and carotenoids, egg yolks of the HIM group were richer (P< 0.05) in γ-tocopherol (+66.7%), lutein (+75.5%),
β-carotene (+73.7%) and total carotenoids (+42.8%) than those of the control group (Table 5).
Fatty acid composition of SBM and HIM yolks is shown in Table 6. Globally, yolks of both groups were extremely rich in 18:1n-9, 16:0, 18:0 (stearic acid), 18:2n-6, 18:1n-7 and 16:1n-7 that accounted for around 73% of total FAs. Inter-estingly, diet significantly affected all FAs except oleic acid, the main constituent of the yolk lipid fraction. Significantly higher contents of LA (+14.4%) and 18:0 (+4.4%) were found in HIM than SBM yolks, whereas HIM yolks were poorer in 16:0 and 16:1n-7 than SBM yolks, with a reduction of 4.1% and 17.5%, respectively. The differences in 18:2n-6 and 16:1n-7 seemed to determine the significant difference in monounsaturated fatty acids and PUFA n-6 fractions found depleted of 2.2% and 6.7%, respectively, in HIM yolks compared with SBM ones. Regarding the products of desaturase/elongase enzyme activity involved in the conver-sion of LA, no differences in 18:3n-6 or 20:4n-6 contents were found between groups, but significantly less 22:5n-6
(−35.1%), that is the final product of this bioconversion, was recorded in HIM than SBM. At the same time, none of the elongation and desaturation products of 18:3n-3, such as 20:5n-3 (eicosapentaenoic acid) and C22:6n-3 (docosahexa-enoic acid), was significantly affected, being stable below 2% of total FAs. Finally, cholesterol was about 11.7% lower in HIM yolks: we recorded 21.92 and 24.84 g/100 g of whole egg in HIM and SBM yolks, respectively, corresponding to 44.98 and 42.13 g/100 g of total lipids.
Discussion
Little information on the effects of insect meal, especially derived from black soldierflies, in the diets of laying hens are available in literature. Most of the experiments published focused on laying performance, weights of egg components and colour, but rarely on egg FA and chemical composition (Agunbiade et al., 2007; Amaoet al., 2010; Makkar et al., 2014; Al-Qazzazet al., 2016). Many studies remark that meal made from larvae ofH. illucens(Al-Qazzazet al., 2016) and larvae of the mothCirina forda(Amaoet al., 2010) can be a good source of protein at different substitution levels (up to 75% using the latter larvae) in the feed of laying hens, without any adverse effect on yolk weight. On the other hand, albu-men weight is reported to be significantly reduced by 50%
C. forda meal substitution, whereas only a slight decrease was obtained with total replacement (Amaoet al., 2010). The present results confirmed that albumen is the egg compart-ment most susceptible to the new protein sources.
Table 3Morphological and physical characteristics and pH of eggs from hens fed soya bean meal diet (SBM) and H. illucens larva meal diet (HIM)
SBM HIM RMSE P-value
Weight (g) Egg 62.67 59.56 5.316 Ns Shell 8.10 7.92 0.762 Ns Yolk 14.80 14.82 1.486 Ns Albumen 39.09a 35.98b 3.936 * Yolk/albumen 0.38 0.42 0.045 *
Percentage on total weight (%)
Shell 12.95 13.3 0.951 Ns Yolk 23.64b 24.95a 1.901 * Albumen 62.33a 60.32b 2.411 * Yolk diameter (cm) 2.87 2.84 0.184 Ns Yolk colour L* 57.83 55.56 4.758 Ns a* 1.36B 5.63A 1.525 *** b* 24.92 27.40 5.106 Ns Chroma 25.00 28.02 5.086 Ns Hue 86.76A 78.01B 3.425 *** pH 6.07 6.10 0.081 Ns
Values are given as average with RMSE (root mean square error).
Ns= not significant.
a,bValues with different superscripts in the same row differ significantly for
P< 0.05 (*).
A,BValues with different superscripts in the same row differ significantly for
P< 0.001 (***).
Table 2Fatty acid composition (g/100 g total fatty acids), cholesterol (mg/kg meal), tocopherol and carotenoid content (mg/kg meal) of experimental diets with soya bean protein source (control diet, C) and H. illucenslarva protein source (H), and profile of H. illucens larva meal H. illucens larvae C diet H diet Fatty acids 12:0 32.34 1.36 7.89 14:0 7.38 1.63 2.85 16:0 13.09 12.95 12.91 16:1n-7 3.59 0.94 1.53 18:0 3.48 3.23 3.42 18:1n-9 12.02 20.55 18.98 18:1n-7 2.50 3.11 3.22 18:2n-6 7.33 36.58 29.73 18:3n-3 1.56 2.67 2.95 20:0 1.27 1.46 1.39 EPA+ DHA 1.14 1.13 1.10 ΣSFA1 61.46 24.74 32.45 ΣMUFA 22.23 28.64 27.66 ΣPUFA n-6 10.10 39.31 32.44 ΣPUFA n-3 4.66 5.75 5.94
Total fatty acids (g/kg meal) 54.59 29.84 31.12
Total tocopherols 42.72 3.41 2.62 Cholesterol ND ND ND δ-tocopherol 1.35 0.39 0.74 γ-tocopherol 3.20 0.43 0.63 α-tocopherol 38.18 2.60 1.25 Retinol 0 0.23 1.36 Total carotenoids 2.15 1.70 6.83 Lutein 1.15 0.96 4.06 Zeaxanthin 0.96 0.69 2.59 β-carotene 0.04 0.05 0.18
ND= Not detected; EPA = eicosapentaenoic acid; DHA = docosahexaenoic
acid; SFA= saturated fatty acids; MUFA = monounsaturated fatty acids;
PUFA= polyunsaturated fatty acids.
1Σ included 13:0, 14:1n-5, 15:0, 16:1n-9, anteiso-17:0, 17:0, 16:3n-4, 17:1,
18:3n-6, 18:3n-4, 18:4n-3, 20:1n-11, 20:1n-9, 20:2n-6, 20:3n-6, 20:4n-6, 20:3n-3, 20:5n-3, 22:0 and 22:6n-3 which were detected but not reported because they were below 1 g/100 g total fatty acids.
The increase in yolk proportion in eggs from hens fed diet H is simply a consequence of the lower albumen proportion. The effect of insect meal on yolk colour is unclear. Data from previous research indicated no effect (Agunbiadeet al., 2007; Amaoet al., 2010) or slight discoloration when 5% of
H. illucensmeal was added to the diet of an Arabian strain of hen; however, as different colour evaluation methods were used, the data cannot be directly compared. In both studies, the authors explained this pattern by the fact that insects are not plants, and therefore do not contain the carotene or xanthophyllous pigments needed for egg colour. However, the results summarized in Table 3 disagree with thisfinding, as yolks of eggs from the HIM group were significantly redder. Furthermore, our data found a clear explanation and confirmation in the total carotenoid content of insect meal and the diet containing insect meal. HIM yolks were found to be rich inβ-carotene and lutein, pigments responsible for an orange–yellow colour. Both pigments, as well as retinol and zeaxanthin, are transferred intact to the egg against a con-centration gradient, so they can build up to higher levels in the egg than their availability in the feed would predict, as reported by Morenoet al. (2016). Considering the carotenoid composition of the two diets and the yolks, a discrepancy clearly emerged, especially for zeaxanthin which doubled in HIM eggs and increased sevenfold in SBM eggs. Egg yolks are known to contain predominantly lutein and are poor sources of pro-vitamin A molecules, such as β-carotene,
presumably because they are utilized by the hen (Suraiet al., 2001) or efficiently stored in the liver (Morenoet al., 2016). The carotenoid profiles reported in the present study confirm this pattern. Looking at the accumulation patterns, both lutein and zeaxanthin were transported against a concentration gradient in both groups, although HIM eggs seemed to accumulate them less than SBM eggs, suggesting that these carotenoids are assimilated less efficiently from
H. illucens meal than from soya bean meal. Morenoet al. (2016) demonstrated that different mechanisms and contexts (e.g. competition, matrix effects and bioavailability) affect transfer and absorption of carotenoids, and the presence of
α-tocopherol in feed is one of such factor, as reported by Islamet al. (2016). Soya bean meal contains twice as much
α-tocopherol as the insect meal, which could be the reason for higher accumulation in SBM than in HIM eggs since, according to Islam et al. (2016), dietary α-tocopherol enhances the bioavailability of lutein. Nevertheless, in the context of human health, eggs from birds fed withH. illucens
meal prove to be a better source of carotenoids, especially lutein, which play a role in the prevention of cataract and age-related macular degeneration, as well as heart disease and stroke (Ribaya-Mercado and Blumberg, 2004), while improving cognitive function in the elderly (Johnson, 2012). The egg composition results obtained are relatively consistent with those of Shinet al. (2013) in terms of total protein and total lipid contents, both being unaffected by the diet, so a total replacement of soya bean with H. illucens
meal may be feasible. To confirm this assertion, a closer look at composition was considered necessary, so the FA composition of the meals and yolks was analysed. St-Hilaire
et al. (2007) and Oonincxet al. (2015) reported that the lipid content of insects depends largely on their diet and stage of development. Regardless the insect growing substrates, 12:0, 16:0 and 18:1n-9 FAs are reported to be the most abundant in black soldier flies (St-Hilaire et al., 2007; Oonincxet al., 2015) and the present results are in line with thesefindings. The FA composition of the two experimental diets was influenced by their specific ingredients. In parti-cular, soya bean was responsible for the high linoleic acid content of diet C, whereas a higher quantity of maize in diet H may explain the difference between the FA composition of
H. illucenslarvae and feed containing it.
Modifications in the FA composition of egg yolks can be obtained by feeding laying hens diets with different levels Table 4Proximate composition (g/kg) of eggs (yolk and albumen) from hens fed soya bean meal diet (SBM) and H. illucens larva meal diet (HIM)
Yolk Albumen
SBM HIM RMSE P-value SBM HIM RMSE P-value
Water content 493.6 505.2 1.88 Ns 869.6 869.8 0.89 Ns
Ash 18.8 17.8 0.25 Ns 7.4 7.5 0.05 Ns
CP 160.1 156.4 0.77 Ns 111.9 110.8 0.81 Ns
Total lipids 325.8 309.3 5.10 Ns – – – –
Values are given as average and root mean square error (RMSE).
Table 5Tocopherol and carotenoid content (mg/kg yolk) of yolks from eggs of hens fed soya bean meal diet (SBM) and H. illucens larva meal diet (HIM)
SBM HIM RMSE P-value
Total tocopherols 44.3 47.8 1.83 Ns δ-tocopherol 3.9 4.3 0.20 Ns γ-tocopherol 2.4b 4.0a 0.20 * α-tocopherol 38.0 39.6 1.65 Ns Retinol 17.0 16.9 0.67 Ns Total carotenoids 10.5b 15.0a 0.64 * Lutein 4.9b 8.6a 0.45 * Zeaxanthin 5.4 6.0 0.46 Ns β-carotene 0.19b 0.33a 0.01 *
Values are given as average and root mean square error (RSME).
a,bValues in the same row with different superscripts differ significantly for
and types of lipid sources, as indicated by Grobas et al. (2001) and Pálet al. (2002). However, not all the differences in yolk FA profile can be attributed to dietary components and the present results are in line with this concept. First of all, 12:0 drastically decreased in HIM yolks, despite the fact that it was abundant in H meal and the diet containing it. In turn, yolk 16:0 and 18:0 almost doubled compared with dietary content, irrespective of the diet used. It is also known that laying hens have remarkable desaturase and elongase activities that transform 18:2n-6 and 18:3n-3 into their
specific derivatives. According to Grobaset al. (2001), most of the increase in n-6 FAs, especially 20:4n-6 and 22:5n-6, as well as increases in 20:5n-3, 22:5n-3 and 22:6n-3, may be due to small increases in the dietary concentration of 18:2n-6 and 18:3n-3, respectively, which enhance enzyme activities. Hence, considering the lower 18:2n-6 content in diet H, the higher values of the same FA found in HIM with respect to SBM eggs and the significantly lower values of its corres-ponding FAs, 20:3n-6 and 22:5n-6, it seems possible to speculate that the enzyme activity of the HIM group was slightly lower. Again, as found for carotenoids, the overall FA composition of HIM yolks was broadly similar to that of the control group. Finally, it is currently accepted that yolk cholesterol level is largely regulated by endogenous origin and not by cholesterol absorption of dietary origin. In the present study, despite the absence of cholesterol in the two experimental meals, cholesterol was found in eggs from both experimental groups (Figure 1) in a concentration in line with the value (15 g/kg of yolk) proposed by Han et al. (1993), thus confirming that cholesterol level in yolks is primarily derived from a balance between biosynthesis and excretion of cholesterol and cholesterol by-products (Sutton et al., 1984). Interestingly, HIM eggs showed cholesterol depletion in line with the reduction in serum levels of cholesterol in laying hens fedH. illucensas exclusive protein source in the trial conducted by Maronoet al.(2015). In this recent study, hens fed an insect-meal-based diet showed lower serum concentrations of cholesterol, namely 108 mg/dl against 134 mg/dl found in hens fed a soya bean-based diet. This reduction in serum cholesterol seemed to depend on the ability of chitin, a natural constituent of insect exoskeleton, to attract negatively charged bile acids and free FAs (Prajapati and Patel, 2010). Notably, the cholesterol reduction in yolk was much less (−11.7%) than that occurring in serum (−20%), thus confirming the importance of a basal level of cholesterol required for egg formation and embryo development. Never-theless, this reduction could be useful for human nutrition, especially for hypercholesterolemic subjects who need to reduce their dietary cholesterol intake (Shinet al., 2013).
In conclusion, H. illucens larva meal seems suitable for total substitution of soya bean meal in diets of Lohmann
0 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 SBM HIM
Cholesterol content (g /100g whole egg)
*
Figure 1 Cholesterol content (g/100 g whole egg) of eggs of hens fed soya bean meal (SBM) and Hermetia illucens larva meal (HIM). Root mean square error: 1.73, *P< 0.05.
Table 6Fatty acid profiles (g/100 g total fatty acids) of yolks from eggs of hens fed soya bean meal diet (SBM) and H. illucens larva meal diet (HIM)
SBM HIM RMSE P-value
Total fatty acids (g/kg) 305.44 307.58 3.114 Ns
12:0 1.19 1.23 0.166 Ns 14:0 1.70B 2.02A 0.214 *** 14:1n-5 0.64b 0.69a 0.081 * 15:0 0.81 0.80 0.105 Ns 16:0 20.82a 19.96b 1.031 * 16:1n-9 1.17b 1.31a 0.125 * 16:1n-7 3.48A 2.87B 0.371 *** Anteiso-17:0 0.77 0.78 0.104 Ns 17:0 0.87 0.88 0.107 Ns 16:3n-4 0.76 0.76 0.101 Ns 17:1 0.83 0.82 0.101 Ns 18:0 7.07b 7.38a 0.255 * 18:1n-9 30.25 30.09 1.467 Ns 18:1n-7 3.83a 3.53b 0.254 * 18:2n-6 10.24B 11.72A 1.005 *** 18:3n-6 0.84 0.83 0.133 Ns 18:3n-4 0.73 0.72 0.095 Ns 18:3n-3 1.01 1.06 0.090 Ns 18:4n-3 0.69 0.69 0.094 Ns 20:0 1.15 1.14 0.165 Ns 20:1n-9 0.66 0.63 0.065 Ns 20:2n-6 0.67 0.66 0.077 Ns 20:3n-6 0.76 0.74 0.081 Ns 20:4n-6 2.70 2.62 0.021 Ns 20:3n-3 0.57 0.57 0.081 Ns 20:4n-3 0.57 0.56 0.077 Ns 20:5n-3 0.55 0.55 0.076 Ns 22:0 0.61 0.61 0.097 Ns 22:5n-6 0.74A 0.48B 0.096 *** 22:6n-3 1.40 1.40 0.159 Ns ΣSFA 35.58 35.38 0.807 Ns ΣMUFA 41.80a 40.88b 1.296 * ΣPUFA n-6 16.20b 17.29a 1.147 * ΣPUFA n-3 4.93 4.98 0.459 Ns
SFA= saturated fatty acids; MUFA = monounsaturated fatty acids; PUFA =
polyunsaturated fatty acids.
Values are given as average and root mean square error (RMSE).
Fatty acids below 0.5 g/100 g total fatty acid are not reported in the table but
were included in theΣ calculation.
Ns= not significant.
a,bValues with different superscripts in the same row differ significantly for
P< 0.05 (*).
A,BValues with different superscripts in the same row differ significantly for
Brown Classic laying hens. Indeed, yolk composition was not negatively affected by the new protein source and all the analytical parameters analysed showed similar or better performance than in eggs from laying hens fed a diet based on soya bean meal as protein source. Yolks from hens fed
H. illucens-based meal did not differ in overall FA compo-sition, whereas they showed higher carotenoid content and an interesting reduction in cholesterol. In any case, some metabolic ways, such as the accumulation of carotenoid and the desaturase/elongase activities, seemed to be slightly inhibited by dietary inclusion of insect meal, hence the specific role of this protein source on these metabolic patterns of laying hens needs to be further investigated.
Acknowledgement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors acknowledge all the technicians of DISPAA-Section of Animal Science of the University of Firenze who supported the present work with their technical assistance.
References
Agunbiade JA, Adeyemi OA, Ashiru OM, Awojobi HA, Taiwo AA, Oke DB
and Adekunmisi AA 2007. Replacement of fishmeal with maggot meal in
cassava-meal based layer diet. The Journal of Poultry Science 44, 278–282.
Al-Qazzaz MFA, Ismail D, Akit H and Idris LH 2016. Effect of using insect larvae meal as a complete protein source on quality and productivity characteristics of
laying hens. Revista Brasileira de Zootecnia 45, 518–523.
Amao OA, Oladunjoye IO, Togun VA, Olubajo K and Oyaniyi O 2010. Effect
of Westwood (Cirina forda) larva meal on the laying performance and egg
characteristics of laying hen in a tropical environment. International Journal of
Poultry Science 9, 450–454.
Association of Official Analytical Chemists (AOAC) 2012. Official methods of
analysis, 19th edition. AOAC, Washington, DC, USA.
Biasato I, De Marco M, Rotolo L, Renna M, Lussiana C, Dabbou S, Capucchio MT, Biasibetti E, Costa P, Gai F, Pozzo L, Dezzutto D, Bergagna S, Martínez S, Tarantola M, Gasco L and Schiavone A 2016. Effects of dietary Tenebrio molitor meal inclusion in free-range chickens. Journal of Animal Physiology and Animal
Nutrition 100, 1104–1112.
Christie WW 1982. A simple procedure for rapid transmethylation of glycerolipids and cholesteryl ester. Journal of Lipid Research 23, 1072–1075. Commission Internationale de l’Éclairage (CIE) 2004. Colorimetry. Publication n. 15. Bureau Central de la CIE, Vienna, Austria. De Marco M, Martínez S, Hernandez F, Madrid J, Gai F, Rotolo L, Belforti M, Bergero D, Katz H, Dabbou S, Kovitvadhi A, Zoccarato I, Gasco L and Schiavone A 2015. Nutritional value of two
insect larval meals (Tenebrio molitorandHermetia illucens) for broiler chickens:
apparent nutrient digestibility, apparent ileal amino acid digestibility and apparent
metabolizable energy. Animal Feed Science and Technology 209, 211–218.
De Marco M, Martínez S, Hernandez F, Madrid J, Gai F, Rotolo L, Belforti M, Bergero D, Katz H, Dabbou S, Kovitvadhi A, Zoccarato I, Gasco L and
Schiavone A 2015. Nutritional value of two insect larval meals (Tenebrio molitor
andHermetia illucens) for broiler chickens: Apparent nutrient digestibility, apparent ileal amino acid digestibility and apparent metabolizable energy. Animal Feed Science and Technology 209, 211–218.
Folch J, Lees M and Sloane Stanley GH 1957. A simple method for the isolation
and purification of total lipides from animal tissues. Journal of Biological
Chemistry 226, 497–509.
Grobas S, Méndez J, Lázaro R, de Blas C and Mateos GG 2001. Influence of source
and percentage of fat added to diet on performance and fatty acid composition of
egg yolks of two strains of laying hens. Poultry Science 80, 1171–1179.
Gutiérrez GPA, Ruiz RAV and Vélez HM 2004. Analisis composicional,
microbiológico y digestibilidad de la proteína de la harina de larvas deHermetia
illuscens L (Diptera: stratiomyiidae) en Angelópolis-Antioquia, Colombia. Revista Facultad Nacional de Agronomía Medellin 57, 2491–2499.
Han CK, Sung KS, Yoon CS, Lee NH and Kim CS 1993. Effect of dietary lipids on liver, serum and yolk cholesterol contents of laying hens. Asian-Australasian Journal of Animal Science 6, 243–248.
International Eggs Commision (IEC) 2015. Egg Industry Review 2015. Retrieved on 30 April 2017 from http/internationalegg.com/wp-content/uploads/2015/08/ AnnualReviews_2015.pdf
Islam KMS, Khalil M, Männer K, Raila J, Rawel H, Zentek J and Schweigert FJ
2016. Effect of dietaryα-tocopherol on the bioavailability of lutein in laying hen.
Journal of Animal Physiology and Animal Nutrition 100, 868–875.
Johnson EJ 2012. A possible role for lutein and zeaxanthin in cognitive function in the elderly. The American Journal of Clinical Nutrition 96, 1161S–1165S. Leeson S 2010. Nutrition & heath: poultry. Feedstuff September 15, 46–53. Majchrzak D, Fabian E and Elmadfa I 2006. Vitamin A content (retinol and retinyl
esters) in livers of different animals. Food Chemistry 98, 704–710.
Makkar HPS, Tran G, Heuzé V and Ankers P 2014. State-of-the-art on use of insects as animal feed. Animal Feed Science and Technology 197, 1–33. Marono S, Loponte R, Lombardi P, Vassalotti G, Pero ME, Russo F, Gasco L, Parisi G, Piccolo G, Nizza S, Meo CD, Attia YA and Bovera F 2017. Productive
performance and blood profiles of laying hens fed Hermetia illucens larvae meal
as total replacement of soybean meal from 24 to 45 weeks of age. Poultry
Science 96, 1783–1790.
Marono S, Piccolo G, Loponte R, Di Meo C, Attia YA, Nizza A and Bovera F 2015.
In vitro crude protein digestibility ofTenebrio molitorandHermetia illucens
insect meals and its correlation with chemical composition traits. Italian Journal
of Animal Science 14, 338–343.
Miranda JM, Anton X, Redondo-Valbuena C, Roca-Saavedra P, Rodriguez JA, Lamas A, Franco CM and Cepeda A 2015. Egg and egg-derived foods: effects on
human health and use as functional foods. Nutrients 7, 706–729.
Moreno JA, Díaz-Gómez J, Nogareda C, Angulo E, Sandmann G, Portero-Otin M, Serrano JCE, Twyman RM, Capell T, Zhu C and Christou P 2016. The distribution
of carotenoids in hens fed on biofortified maize is influenced by feed
compo-sition, absorption, resource allocation and storage. Scientific Reports 6, 35346. National Research Council (NRC) 1994. Nutrient requirements of poultry, 9th revised edition. National Academy Press, Washington, DC, USA.
Oonincx DGAB, van Broekhoven S, van Huis A and van Loon JJA 2015. Feed conversion, survival and development, and composition of four insect species on diets composed of food by-products. PLoS One 10, e0144601.
Pál L, Dublecz K, Husvéeth F, Wágner L, Bartos Á and Kovács G 2002. Effect of dietary fats and vitamin E on fatty acid composition, vitamin A and E content and oxidative stability of egg yolk. Archiv für Geflügelkunde 66, 251–257. Prajapati BG and Patel RP 2010. Nutrition, dietary supplements and herbal medicines: A safest approach for obesity. Research Journal of Pharmaceutical,
Biological and Chemical Science 1, 39–45.
Prates JAM, Quaresma MAG, Bessa RJB, Fontes CMA and Alfaia CMM 2006. Simultaneous HPLC quantification of total cholesterol, tocopherols and
β-caroteneee in Barrosã-PDO veal. Food Chemistry 94, 469–477.
Ribaya-Mercado JD and Blumberg JB 2004. Lutein and zeaxanthin and their potential roles in disease prevention. Journal of the American College of
Nutrition 23, 567S–587S.
SAS 2004. SAS/STAT user’s guide (Release 9.1). SAS Institute Inc., Cary, NC, USA.
Shin JY, Xun P, Nakamura Y and He K 2013. Egg consumption in relation to risk of cardiovascular disease and diabetes: a systematic review and meta-analysis.
The American Journal of Clinical Nutrition 98, 146–159.
St-Hilaire S, Cranfill K, McGuire MA, Mosley EE, Tomberlin JK, Newton L, Sealey W, Sheppard C and Irving S 2007. Fish offal recycling by the black soldier fly produces a foodstuff high in omega-3 fatty acids. Journal of the World
Aquaculture Society 38, 309–313.
Surai PF, Speake BK and Sparks NHC 2001. Carotenoids in avian nutrition and embryonic development. 1. Absorbtion, availability, and levels in plasma and
egg yolk. Journal of Poultry Science 38, 1–27.
Sutton CD, Muir WM and Mitchell GE Jr 1984. Cholesterol metabolism in the
laying hen as influenced by dietary cholesterol, caloric intake, and genotype 1.
Poultry Science 63, 972–980.
van Huis A, Van Itterbeeck J, Klunder H, Mertens E, Halloran A, Muir G and Vantomme P 2013. Edible insects. Future prospects for food and feed security. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy.
